This chapter provides an overview of advanced in vitro breeding technologies for donkeys and mules, focusing on the latest advancements and their potential applications in equid reproduction and conservation. We begin by discussing ovum pick-up (OPU) and the techniques for in vitro oocyte maturation and vitrification, highlighting their critical roles in enhancing the efficiency of advanced reproductive technologies (ARTs). The development of intracytoplasmic sperm injection (ICSI) is explored in depth, including the first successful production of domestic donkey and mule blastocysts in laboratory settings. These recent breakthroughs mark a significant milestone in long-ear reproductive research and opens new possibilities for improving breeding programs. Challenges and advancements associated with in vitro fertilization (IVF) in equids are covered, noting the recent more reliable IVF protocols for horses and their future application in other equids. Cloning is addressed with reference to the first cloned equid, a cloned mule, Idaho Gem, and its implications for research, competition, and conservation of other endangered equids. Finally, the role of newly developed technologies in reproductive medicine associated with stem cells is examined with the possibility of creating gametes or embryos in vitro and addressing reproductive challenges in donkeys and mules. In conclusion, this chapter underscores the transformative impact of advanced in vitro breeding technologies on donkeys and mules for addressing reproductive challenges and enhancing genetic diversity.